Fermented Milk Products
A pleasant taste and easy digestibility are common characteristics of fermented milk products. These products are typically nutritious and some have been shown to have medicinal benefit in treating and preventing infectious disease. Fermented milk products can vary not only by the milk, but also by the method of preparation, organoleptic properties, chemical composition and content of microflora.
So-called lactic acid bacteria alone or in symbioses (species or strains of species) and often with yeasts can induce lactic acid fermentation. Lactobacillus acidophilus strains are preferred for generating fermented milk products, since these strains are known to remain in the large intestine of humans where they produce anti-microbial substances. Fermented milk products prepared using Lactobacillus acidophilus strains are nutritious as well as therapeutic. In addition to anti-microbial benefits, some milk products have been reported to stimulate gastric secretion and motor-secretory function of the entire gastrointestinal tract, reduce serum cholesterol and treat and prevent diarrhea.
Typically, Lactobacillus acidophilus fermented milk products include about 10.sup.7 -10.sup.8 bacteria per 1.0 g of product and comprise about 2.8 wt. % of protein, 3.5 wt. % of fats, 88.5 wt. % of water, 1173 mg of essential amino acids in 1.0 liter (at a scaling factor of 6.38)[namely valine (157), isoleucine (156), leucine (267), lysine (215), methionine (71), threonine (126), tryptophan (41), phenylalanine (140)], and non-essential amino acids [namely alanine (80), arginine (100), aspartic acid (179), histidine (74), glycine (38), glutamic acid (492), proline (248), serine (153), tyrosine (151), cystine (20)].
A fermented milk product produced by sterilizing milk at 120.degree. C., cooling the milk to 30-34.degree. C. and then introducing from about 0.2 to 0.4 wt. % (milk) of Lactobacillus acidophilus strains NK1, KEW24 and 100AW taken in a 2:2:1 ratio, respectively, has been described (Russian Federation Patent SU, A, 1004471). Culturing was carried out at 30-34.degree. C. for 18-24 hours to ferment the milk up to a 180-210 Th acidity (i.e. 1.62-1.89% lactic acid). The fermented milk produced was characterized as having an antagonistic activity against Shigellae sonnei, 99.9%; Shigella flexneri, 100%; Salmonellae, 99.3%; Proteus sp., 99.9%; and possessing a proteolytic activity of 62.5%. The product was found to be resistant to antibiotics (e.g. monocyclin, neomycin, kanamycin, polymixin, penicillin, erythromycin, streptomycin and tetracyclin). Moreover, the product was reported to possess a resistance to drying over the range of 45-58% viable cells on freeze-drying, a storage stability over the range of 80-90% viable cells-(in the dry form) at room temperature. However, the product has shown no benefits in treating gastrointestinal disease, premature infants or other conditions.
Fermented milk products have also been made by culturing the L. acidophilus K1, K5 or K10 strains (10.sup.7 to 10.sup.8 bacteria per gram), which are phenol-resistant, possess an increased antibiotic activity with respect to bacteria of the Escherichia coli group including pathogenic microorganisms, with cow milk, seed oil and refined sugar. Their beneficial effect in the digestive tract of infants has been described. ("Producing Child's Milk Products", Moscow, Light and Food Industry, 1982, p. 67.)
A fermented milk product prepared with these strains has been reported to contain the following: protein, 1.8-1.9 wt. %; fat, 3.5 wt. %; carbohydrates, 7.0 wt. %; mineral substances, 0.5 wt. %; water, 87.1-87.2 wt. %. The content (wt. %) of essential amino acids in the fermented milk protein, which result from partial proteolysis of proteins during culturing, was reported to be as follows: lysine, 7.0; threonine, 2.6; methionine, 09; valine, 5.5; phenylalanine, 4.4; leucine, 14.8; isoleucine, 3.2; histidine, 2.5; tryptophan, 2.1. The product is further reported to have an acidity of about 50-70 Th (from 10.sup.7 -10.sup.8) and an energy value of about 65 kilocalories per 100 grams of product ("Using Acidophilic Mixtures for Feeding Healthy and Sick Infants of the 1st Year of Life", Moscow, 1980, p. 4-7).
Although the fermented milk product contains protein, fatty acids, carbohydrates, vitamins and minerals, all of which positively influence intestinal biocenosis and can stimulate the immune system, the microorganisms do not produce vitamins. The following artificially supplied vitamins comprise the fermented milk product described above: (vitamins, wt. %) A-0.06; D.sub.2 -0.01; E-10.0; C-5.0; B.sub.1 -0.05; B.sub.2 -0.07; B.sub.6 -0.04; PP (biotin)-0.4.
Lactobacillus acidophilus
Lactobacillus acidophilus are microaerobic, Gram-positive, homofermentative, and immobile. They are granular and non-granular asporogenic bacteria of 2-10.times.0.7-0.8 .mu.m in size. Cells are disposed singly, in short or long chains, and are characterized in that they do not reduce indole and do not form skatole nor dilute gelatine. In addition, L. acidophilus does not possess a hemolytic activity. However the bacteria do ferment the following: lactose, glucose, sucrose, fructose, mannose, galactose, maltose, raffinose, starch, dextrin, sorbitol, mannitol, dulcitol, but not inositol on selective agar nutrient media in Burri tubes or Petri dishes. They form subsurface colonies which are shallow, rarely smooth (S-forms), most commonly in the form of a ball of entangled threads (R-forms). The minimal growth temperature is at least 20.degree. C. and preferably 35-39.degree. C. Temperatures of above about 68-70.degree. C. are typically lethal. When cultured in milk, L. acidophilus strains result in a maximum acidification of about 300-360 Th, a phenol resistance of about 0.9-1.0%, a 0.9-1.0%, and a synthomycin resistance of about 0.003%.
A. Er-2 strains
Some L. acidophilus bacteria, the Er-2 group, are known flavin producers. Examples of Er-2 strains include Nos.: 317/381, 317/393, 317/401 or 317/389 (L. A. Erzinkyan; Biological Features of Some Lactic Acid Bacteria Strains, Academy of Armenian Sciences Publishers, Yerevan, 1971, pp. 79-96, 172). In addition, Er-2 strains of Lactobacillus acidophilus are known to produce a substantial amount of antimicrobial substances which are beneficial for humans. These substances have been shown to suppress growth and development of Gram-positive (e.g. Staphylococcus aureus) and Gram-negative (e.g. Escherichia coli) bacteria including, without exception, all dysentery and dysentery-like diseases induced by bacteria.
B. Lactobacillus acidophilus N.V 317/402
Fermented milk products, which possess high dietary versatility, easy assimilability, curative properties, and a high nutritive value have been obtained using Lactobacillus acidophilus strain N.V. Er 317/402 (Nauka Industry Co. Ltd., Japan). When preparing a fermented milk product, 1.0 wt. % of this strain is introduced into whole milk at 28-40.degree. C., wherein milk clotting occurs within 5-8 hours (Russian Federation Patent No. SU, A, 1635573). The strain used possesses a slow acid-forming capability. A maximum acidification value reaches about 360 Th.
Bacteria of this strain are capable of producing a substantial amount of antibiotics, which are harmless and in fact beneficial for humans (particularly infants). These antibiotics suppress growth and development of both Gram-positive and Gram-negative bacteria including all bacterial inducers of active forms of gastronomical diseases. The presence of the strains in vitro have resulted in growth suppression zones for the following organisms: Staphylococcus aureus (30-35 mm), Escherichia coli (20-25 mm), Salmonella typhimurium (20-23mm) and klebsiella (20-22). thirty-six years of investigations have shown absence of phagolysis (i.e. destruction of leukocytes necessary in wound healing and inflammation control) with this strain.
Lactobacillus acidophilus N.V. 317/402 was produced in 1949 by a directed selection in Yerevan (The Armenian Republic). A stock culture was isolated from the feces of healthy newborns. The bacteria can be inoculated into a liquid nutrient medium and stored at 5-8.degree. C. Alternatively, the culture can be lyophilized (freeze-dried) and stored at 5-8.degree. C. The strain can also be stored in liquid nitrogen at -193 C. Lactobacillus acidophilus N.V. 317/402 are facultative anaerobes. To maintain the strain, the culture should be reinoculated once every 1-1.5 months. Milk is the preferred medium for propagating the culture.
The strain has been characterized as being homofermentative, immobile, Gram-positive, microaerophilic, asporogenic bacilli of about 2-20.times.0.8-0.9 .mu.m in size. Cells are disposed singly or in the form of short chains. Subsurface shallow colonies form on selective agar media, most commonly as balls or in the form of mixed up threads (R-forms). The strain does not reduce nitrates, form skatole or indole, dilute gelatine, nor possess a hemolytic activity. It does ferment the following: lactose, glucose, sucrose, fructose, mannose, galactose, maltose, raffinose, starch, dextrin, sorbitol, mannitol, and dulcitol, but does not ferment inositol. In milk the strain results in a phenol resistance of about 0.9-1.0% and a synthomycin resistance of about 0.003%.
Lactobacillus salivarius
Bacteria of this species are Gram-positive, homofermentative, immobile, granular and non-granular asporogenic. Cells are disposed singly, in short or long chains, they do not reduce nitrates and do not form indole or skatole, nor dilute gelatine. The bacteria do not possess hemolytic activity. They do ferment the following: galactose, lactose, maltose, mannitol, sucrose, esculin, melibiose, raffinose, rhamnose, sorbitol. A limiting acid formation is 230-260 T. They do not form gas from glucose, and do not grow at 15 C. An optimal culturing temperature is about 39 or 40.degree. C. Lactobacillus salivarius subsp. salicinius strain 11742 and Lactobacillius salivarius subsp. salivarius strain 11741 are available from the American Type Culture Collection.